Plasma Acetone Metabolism in the Fasting Human

G. A. REICHARD, JR., A. C. HAFF, C. L. SKUTCHES, P. PAUL, C. P. HOLROYDE,and0. E. OWEN,Department of Research, Lankenau Hospital, Philadelphia,Petnnsylvania 19151, Department of Medicine and the General Clinical ResearchCenter, Temple University Health Sciences Center, Philadelphia, Pennsylvania19140

A B S T RA C T The metabolism of acetone was stud-ied in lean and obese humans during starvation keto-sis. Acetone concentrations in plasma, urine, andbreath; and rates of endogenous production, elimina-tion in breath and urine, and in vivo metabolism weredetermined. There was a direct relationship betweenplasma acetone turnover (20-77 ,Umol/m2 per min) andconcenitration (0.19-1.68 mM). Breath and urinary ex-cretion of acetone accounted for a 2-30% of the endog-enous production rate, and in vivo metabolism ac-counted for the remainder. Plasma acetone oxidationaccounted for -60% of the production rate in 3-d fastedsubjects and about 25% of the production rate in 21-dfasted subjects. About 1-2% of the total CO2productionwas derived from plasma acetone oxidation and wasnot related to the plasma concentration or productionrate. Radioactivity from [14C]acetone was not detectedin plasma free fatty acids, acetoacetate, ,-hydroxy-butyrate, or other anionic compounds, but was presentin plasma glucose, lipids, and proteins. If glucose syn-thesis from acetone is possible in humans, this processcould account for 11% of the glucose production rateand 59% of the acetone production rate in 21-d fastedsubjects. During maximum acetonemia, acetone pro-ductioni from acetoacetate could account for 37% of theanticipated acetoacetate production, which implies thata significant fraction of the latter compound does notundergo immediate terminal oxidation.

INTRODUCTION

The extent to which acetone quantitatively contributesto starvation-induced and diabetic ketonemia has re-mained controversial for many years. With recently de-veloped methods for acetone determination in blood,breath, and urine, coupled with isotope tracer tech-

Address reprint requests to Dr. George A. Reichard, Jr., Lan-kenau Hospital.

Received for publication 11 August 1978 and in revisedform 11 December 1978.

niques, we have measured rates of endogenous acetoneproduction, breath, and urinary excretion and conver-sion to other biological compounds during starvation-induced ketonemia in the human. In addition, studieswere also done to determine the relationship amongplasma acetone, acetoacetate (AcAc),1 and J8-hydroxy-butyrate (p-OHB) specific activities during the con-tinuous infusion of [3-14C]AcAc.

METHODS

Subjects. Clinical data for the 15 volunteer subjects whoparticipated in these studies are shown in Table I. The sub-jects were of both sexes and ranged in age from 22 to 52 yr.Before admission, the purpose and potential risks of the pro-cedures were discussed with each subject, and informed writ-ten consent was obtained. All subjects had normal hemograms,urinalyses, electrocardiograms, chest radiographs, and sequen-tial multiple analyzer (SMA) 12/60 profiles. 2-h postprandialblood glucose concentrations were also normal. Duringstarvation, daily intake consisted of one multivitamin capsule(Unicap, The Upjohn Co., Kalamazoo, Mich.) and at least1,500 ml of water.

[2-l4C]acetone preparation and administration, sample col-lection, and analyses. [2-14C]acetone (Amersham Corp.,Arlington Heights, Ill.), with a 26 mCi/mmol sp act, was usedin all studies. It was dissolved in ice-cold isotonic saline andsterilized by passage through a 0.22-,um Swinnex filter unit(Millipore Corp., Bedford, Mass.). This primary [14C]acetonesolution contained 25 ,.LCi/ml.

On the day of the isotope study, the subject was at rest on abed in a special room equipped with an efficient ventilationsystem to exhaust exhaled '4CO2 and to maintain a normalroom air-gas content. The subject voided before the study,and urine was collected throughout and at the close of theexperimental period and stored on ice in a closed containeruntil the appropriate analyses were performed. An antecubitalvenous catheter was inserted in each arm for administrationof the ['4C]acetone and for collection of blood samples.

Immediately before use, an aliquot of the primary [P4C]ace-tone solution was diluted in a glass syringe with ice-cold iso-tonic saline, a sample was removed for radiochemical assay,and the remaining solution was rapidly administered intra-

I Abbreviations used in this paper: AcAc, acetoacetate; ,3-OHB, /8-hydroxybutyrate.

619J. Clini. Inivest. (D The Amitericatn Society for Clinical Investigation, Inc. 0021-9738/79/04/0619/08 $1.00Volume 63 April 1979 619-626

TABLE IClinical Data*

Weight on day Body surfaceHeight of study area

cm kg m2Nonobese, 3-d

fasted(n = 6)

Mean+SEM 179±5 72.3±4.6 1.91±0.09(range) (165-193) (59.5-87.3) (1.65-2.12)

Obese, 3-dfasted(n = 6)

Mean±SEM 164±3 108.7±7.4 2.11±0.07(range) (160-177) (82.7-137.8) (1.90-2.41)

Obese, 21-dfasted(n = 3)

Mean±SEM 168±8 124.1±13.9 2.29±0.18(range) (160-183) (101.9- 149.8) (2.04-2.64)

* On admission, nonobese subjects were -7-+16% and theobese subjects +38-+155% of ideal body weight based onMetropolitan Life Insurance Tables, 1959. n is the numberof subjects in each group from which the mean was obtained.The range of values in each group is shown in parentheses.

venously. The quantity of [14C]acetone administered was26.6-70.7 ,uCi, 1.01-2.72 ,umol.

Beginning 1 h after ['4C]acetone administration, and athourly intervals thereafter, heparinized blood samples werewithdrawn and plasma obtained by centrifugation at 4°C.

The method to determine plasma acetone concentration andradioactivity was based on the use of plasma to avoid possibletime-related spontaneous decarboxylation of AcAc, whichcould occur during preparation of protein-free filtrates. AcAc,originally present in plasma, was first converted to /3-OHB,using /8-OHB dehydrogenase, after vyhich acetone was iso-lated from the reaction mixture by distillation. This methodhas been published in detail (1) and will be briefly outlinedas follows. The conversion of AcAc to /8-OHB'was carriedout in a distilling flask connected to an all-glass apparatus toavoid possible loss of acetone during the incubation period.The reaction mixture consisted of plasma (containing not morethan 10 ,umol AcAc), 200 ,umol phosphate buffer (pH 7.0), 45,umol NADH, 3 U ,8-OHB dehydrogenase, and distilled waterto make a final vol of 50 ml. After a 2-h incubation period atroom temperature, acetone was removed by distillation (1).30 ,umol of carrier AcAc was added to an aliquot of the distil-late and treated as previously described to obtain Denig&'ssalt of acetone for counting (2). Acetone concentration in thedistillate was determined by an automated procedure (1) basedon the alkaline-salicylaldehyde method of Procos (3). Radio-activity in AcAc was obtained as the difference betweenplasma samples treated as above and equivalent plasma sam-ples in which the conversion of AcAc to ,/-OHB was not done.,8-OHB radioactivity was obtained from the residue remainingafter distillation of acetone and AcAc by converting j3-OHBto AcAc enzymatically and repeating the distillation.

Aqueous, plasma, and whole blood standards containingknown amounts of [2-14C]acetone, [3-'4C]AcAc, and [3-14C]-_3-OHB (as the D[-] isomer) were subjected to the completeprocedure, and 97- 101% of added radioactivity was routinelyrecovered in the expected fractions.

Aliquots of.pooled urine were assayed for acetone concen-tration and radioactivity in acetone, AcAc, and ,1-OHB by theprocedure described for plasma. Appropriate modificationsin urine sample size or the reaction mixture components weremade to not exceed the capacity of the system for convertingAcAc to ,8-OHB.

AcAc and ,8-OHB concentrations in plasma and urine weredetermined with enzymatic techniques (4). AcAc concentra-tions and acetone concentrations and radioactivity in plasmaand urine were determined on the day of study.

The determination of acetone concentration in expired airpresented special problems because of significant loss of thiscompound when air collections were made by usual tech-niques employing Douglas bags. A special device, consistingof a calibrated 2-liter glass suction flask equipped with a 1.5-cmdiameter glass inlet tube, was used. The side arm of the suctionflask was fitted with a rubber septum through which samplesof collected air were removed with Pressure-Lok gas syringes(Supelco, Inc., Bellefonte, Pa.). Breath collections were madeby having the subject breathe through the inlet tqbe at a nor-mal rate and volume for 3 min, after which the collecting devicewas closed. Acetone concentration was determined on 100- to 500-,ul air samples with a gas chromatograph equipped with a flameionization detector and a 183 cm x 2-mmglass column packedwith Carbopack c/0.1% SP-1000 (Supelco, Inc.). Acetone con-centration was determined from peak height by comparisonwith gaseous acetone standards prepared in calibrated 2-litersuction flasks. Acetone output in breath was calculated fromthe breath acetone concentration and the minute-volume,measured with the Douglas bag technique.

Respiratory gas samples were also obtained simultaneouslywith each blood sample for analysis of 02, C02, and '4CO2content (5). Preliminary studies with prepared air samplescontaining [14Clacetone showed no contamination of the CO2trap of the Fredrickson and Ono system (6) which is used inour laboratory to determine CO2 specific activity.

Incorporation of radioactivity into plasma glucose and an-ionic compounds, including lactate, was estimated with meth-ods previously described (7). Briefly, this procedure consistedof the preparation of a protein-free filtrate of plasma (8), theelimination of[14C]acetone from the filtrate by partial evapora-tion with heat under a stream of nitrogen, and separation ofneutral from charged molecules with an Amberlite MB-3(Rohm and Haas Co., Philadelphia, Pa.) resin column. Glucosein the column eluate was oxidized to gluconic acid withglucose oxidase and isolated for counting purposes with asecond column of Amberlite CG-400 resin. Anionic com-pounds were eluted from the MB-3 column for counting with0.5 MNaCl.

Total radioactivity in plasma and urine was determined withBray's solution (9), in plasma free fatty acids by a pre-viously described method (5), in plasma total lipids afterextraction by the method of Folch et al. (10), and in plasmaproteins after precipitation with 6%perchloric acid. The proteinprecipitate was washed twice with 6%perchloric acid, extractedby the method of Folch et al. (10), and the delipidated pro-tein was suspended and counted in Scintisol (Isolab, Inc.,Akron, Ohio).

Calculations. After [14C]acetone administration, the lineardecline in plasma acetone specific activity with time, whichwas observed in all subjects, is compatible with first-orderreaction-rate kinetics. During each study period, the plasmaacetone concentration remained constant, indicating steady-state conditions were present. From the known quantity andspecific activity of injected [14C]acetone and the extrapolatedzero time plasma acetone specific activity, the acetone poolwas calculated. The fractional replacement rate of acetone (k)was obtained with the simplified equation k = 0.693/t112, wheret1/2 was determined from the slope of the specific activity curve.

620 Reichard, Haff, Skutches, Paul, Holroyde, and Owen

The acetone turnover rate was calculated as the product ofthe acetone pool and fractional replacement rate. The ap-parent acetone space was calculated from the acetone pooland the average plasma acetope concentration with the as-sumption that this concentration was uniformly presentthroughout the entire space.

Studies with [3-14C]AcAc. Two nonobese subjects re-ceived a primed continuous infusion of [3-14C]AcAc for 6 hinstead of single injections of [14C]acetone. The methods forpreparation and administration of the [P4C]AcAc have beenpublished (2). During the infusion period, hourly blood sam-ples were obtained, and plasma acetone, AcAc, and 18-OHBconcentrations and specific activities were determined asdescribed.

RESULTS

Venous plasma ketone-body concentrations. TableII shows the mean venous plasma ketone-body con-centrations in each group of subjects during the isotope-tracer study periods. After a 3-d fast, plasma acetone,AcAc, and ,8-OHB concentrations in the nonobese sub-jects were significantly greater than in the obese sub-jects. Plasma ketone-body concentratiohs in 21-d fastedobese subjects were also significantly greater than in3-d fasted obese subjects. In some subjects, plasmaacetone concentrations were close to or greater thanplasma AcAc concentrations. Plasma AcAc and f3-OHBconcentrations were similar to those previously re-ported for subjects of similar body weights and dura-tion of fast (11).

Specific activity of plasma acetone, glucose, and

TABLE IIVenous Plasma Ketone-Body Concentrations*

Acetone AcAc P-OHB

mMNonobese, 3-d

fasted(n = 6)

Mean±SEM 0.76±0.16 1.20±0.07 3.57+0.37(range) (0.46-1.44) (0.97-1.51) (2.80-5.05)

Obese, 3-dfasted(n = 6)

Mean±SEM 0.29±0.04t 0.70+0.08t 2.16±0.51§(range) (0.19-0.47) (0.51-1.09 (1.20-4.09)

Obese, 21-dfasted(n = 3)

Mean±SEM 1.37±0.19 1.83±0.29 6.45±0.92(range) (1.03-1.68) (1.44-2.40) (4.71-7.83)

* Mean±SEMvalues shown are of individual mean valuesobtained during each study period. The range of individualmeans is in patentheses. n is the number of subjects ineach group.t P 5 0.01 vs. noriobese, 3-d fasted and obese, 21-d fasted.§ P c 0.05 vs. nonobese, 3-d fasted and obese, 21-d fasted.

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FIGURE 1 Time-course changes in the specific activity ofplasma acetone, glucose, and breath CO2. The steady-stateplasma acetone concentration is shown at the bottom ofthe figure. The obese subject had fasted for 21 d. Conc.,concentration.

breath CO,. Fig, 1 shows the time-course changesin the specific activities of plasma acetone, glucose,and breath CO, after the pulse administration of ['4G]-acetone to a 21-d fasted obese subject. During the 6-hstudy period, the plasma acetone concentration wasconstant while the specific activity slowly declined ata linear rate. The specific activities of plasma glucoseand breath CO, rose during the study neriod, but at agradually decreasing rate. No detectable radioactivitywas found in plasma AcAc, 18-OHB, free fatty acids,or anionic compounds in this or any subject of thesestudies.

Acetone pools, spaces, and fractional replacementrates. Acetone pools ranged from 5.91 to 56.4mmol/m2. As shown in the top panel of Fig. 2, therewas a direct linear relationship between plasma ace-tone concentrations and acetone pools. The apparentacetone space, expressed as precent body weight,averaged 76.3% (62.1-93.6%) in 3-d fasted nonobesesubjects, 62.5% (45.4-86.9%) in 3-d fasted obese sub-jects, and 58.9% (56.1-61.5%) in 21-d fasted obesesubjects. Although of limited physiological significance,these results suggest that acetone was distributed in aspace that exceeded total body water. The hydrophylicand lipophylic nature of acetone may be responsible

Acetone Metabolism in the Human 621

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r=0.98

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FIGURE 2 Relationship between plasma acetone concentra-tion and acetone pool (top panel) or fractional replacementrate (bottom panel). A significant correlation between concen-tration and pool (r = 0.98, P < 0.001) or concentration andfractional replacement rate (r = 0.67, P < 0.01) was obtained.K%min-', fractional replacement rate.

for this. The bottom panel of Fig. 2 shows an inverserelationship between the fractional replacementrates of the acetone pool and the plasma acetoneconcentrations.

Acetone turnover rates. Fig. 3 shows the relation-ship between the acetone turnover rates, expressedas micromoles per square meter per minute, andthe plasma acetone concentrations. Values were nor;malized to square meters of body surface areabecause of the wide range of body weights ofthe subjects. Although not shown, a similar, highly sig-nificant correlation (r = 0.84, P < 0.001) was also ob-tained when the acetone turnover rate was expressed asmicromoles per minute. The mean plasma acetone turn-over rate in the 3-d fasted nonobese subjects was 51(20-69) ,umol/m2 per min, in the 3-d fasted obese sub-jects was 31 (20-41) /Amol/m2 per min, and in the 21-dfasted obese subjects was 61 (51-77) ,tumol/m2 per min.The difference between 3- and 21-d fasted obese sub-jects was statistically significant (P < 0.002).

Urine and breath acetone. The relationship be-tween plasma acetone concentrations and urine orbreath concentrations are shown in Fig. 4. Chemicallydetermined urine acetone concentrations, shown byclosed circles in the left panel of Fig. 4, were obtainedfrom aliquots of pooled urine collected during the 6-hacetone turnover studies, and the plasma acetone con-centrations are average values observed in each sub-

ject during the same time period. The chemically de-termined urine acetone concentrations were 10-foldgreater than plasma concentrations. However, urineacetone specific activities were considerably less thanplasma acetone specific activities, indicating thatplasma acetone was not the sole source of urine ace-tone. At least in part, the lower urine acetone specificactivity may have been a result of formation of acetonefrom unlabeled AcAc in urine during the 6-h collectionperiod. To estimate the fraction of urine acetone de-rived from plasma, the ratio of urine acetone specificactivity to plasma acetone specific activity at the mid-point of each study period was determined. On the basisof these calculations, -8-29% of urine acetonewas derived from plasma. Urine acetone concentration,calculated from these percentages and the chemicallydetermined concentrations, are shown as open circlesin the left panel of Fig. 4. Rates of acetone excretionin urine, based on chemically determined and cor-rected urine acetone concentrations are shown in TableIII. For all subjects, plasma acetone clearance rangedfrom 0.8 to 2.3 ml/min, suggesting marked renal re-absorption or back-diffusion of the compound.

The relationship between breath and plasma acetoneconcentrations is shown in the right panel of Fig. 4.The range of breath acetone concentrations observedin our subjects is similar to that previously reportedin fasting humans (12). Below plasma acetone concen-trations of -0.5 mM, ratios of plasma to breath con-centrations were in the range of 500:1-600:1, whereasat higher plasma concentrations, the ratios were some-what smaller, i.e., 350:1-500:1. In the human, after theoral ingestion of acetone, a blood:breath ratio of 330:1at blood concentrations of 0.5-1.2 mMhas beenreported (13, 14).

Metabolic and excretory fate of acetone. Under

w

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FIGURE 3 Relationship between plasma acetone concentra-tion and turnover rate. A significant correlation between con-centration and turnover rate (r = 0.80, P < 0.001) wasobtained.

622 Reichard, Haff, Skutches, Paul, Holroyde, and Owen

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z0LI

wcrco

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5-

4-

3-

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FIGuRE 4 Relationship between plasma acetone concentra-tion and urine concentration (left panel) or breath concentra-tion (right panel). Urine concentrations represented by closedcircles were chemically determined, and those by open circleswere calculated from urine and plasma specific activities (seetext).

steady-state conditions, the acetone turnover rate isequal to its rate of production and disappearance. TableIV shows the percent of the measured acetone produc-tion rate which could be accounted for by breath andurinary excretion and, by difference, other in vivometabolic fates in each subject. In general, the fractionof the acetone production rate accounted for by breathand urinary excretion was directly related to theacetone production rate and plasma concentration.Conversely, the fraction of the acetone production rateaccounted for by in vivo metabolism decreased with

increasing production rates and plasma concentrations.In all subjects, however, in vivo metabolism was themajor mechanism for acetone disposal.2 Similar resultshave also been obtained in studies in rats and humansafter administration of large quantities of nonradioactiveacetone (14, 15).

Incorporation of [14C]acetone radioactivity intoother compounds. After [14C]acetone administration,radioactivity was found in numerous other biologicalcompounds. Cumulative 14C02 excretion during the 6-hturnover study periods in the 3-d fasted nonobese,obese, and 21-d fasted obese subjects accounted for17.4% (10.7-21.3%), 21.5% (12.9-28.7%), and 4.9%(3.1-7.1%), respectively, of the administered [14C]-acetone radioactivity. The lowest cumulative 14CO2 ex-cretion occurred in the most acetonemic subjects andprobably reflects dilution of the administered [14C]ace-tone in larger body acetone pools. In all subjects, therespiratory CO2 specific activity curves were similarto that shown in Fig. 1, i.e., the values observed at6 h were probably close to the maximum which wouldhave been achieved if our studies had been prolonged.With the 6-h values of plasma acetone and respiratoryCO2 specific activities, the percent CO2 derived fromplasma acetone oxidation was calculated from theequation: %= (sp act CO2/sp act acetone + 3) x 100.Values for the 3-d fasted nonobese, obese, and 21-dfasted obese subjects were 1.62% (1.25-1.80%), 1.51%(0.82-2.12%), and 1.11% (0.73-1.33%), respectively.Total respiratory CO2 outputs in these same groupswere 4.58 (3.28-5.69), 4.40 (3.57-5.06), and 4.14 (4.00-4.38) mmol/m2 per min. From the total respiratory CO2outputs and the percent CO2derived from acetone oxi-

2 Preliminary studies in our laboratory have shown that ace-tone excretion through skin can account for no more than0.5% of the production rate per square meter of body surfacearea.

TABLE IIIUrine Acetone Cotncentration, Specific Activity Ratio, and Excretion

SpecificChemical Urinary activity Corrected Corrected

concentration excretion ratio concentration excretion

.rnMol/ml /Lmollmifl S 'mUo0/ml umol/minNonobese, 3-d fasted (n = 4)

Mean+SEM 7.6+0.8 6.4±1.2 17+3 1.2+0.3 1.2+0.4(range) (5.5-9.5) (4.2-9.2) (8-23) (0.6-1.8) (0.4-2.1)

Obese, 3-d fasted (nt = 6)Mean±SEM 3.5±0.6 3.1±0.5 14±3 0.4±0.04 0.4±0.06(range) (1.9-4.9) (1.5-5.1) (8-29) (0.3-0.6) (0.2-0.6)

Obese, 21-d fasted (n = 3)Mean+SEM 14.6±3.2 8.1+1.5 18+1 2.6±0.6 1.7±0.1(range) (9.6-20.6) (5.1-9.7) (16-20) (1.9-3.7) (1.5-1.9)

n is the number of subjects in each group from which the mean was obtained. The rangevalues in each group is shown in parentheses.

Acetone Metabolism in the Humant 623

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8-

6-

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TABLE IVAcetone Disposal Mechanisms

Percentage of acetone turnover rate accounted for as

OtherBreath Urinary metabolic

excretion excretion fates

Nonobese, 3-dfasted(n = 4)

Mean+SEM 14.7±2.3 1.4±0.5 83.9±2.6(range) (10.3-19.2) (0.4-2.7) (79.1-89.3)

Obese, 3-dfasted(n = 6)

Mean±SEM 5.3±1.8 0.6±0.1 94.1±1.8(range) (1.8-12.1) (0.3-0.8) (87.1-97.8)

Obese, 21-dfasted(n = 3)

Mean±SEM 25.2±3.6 1.3±0.2 73.5±3.4(range) (18.1-28.9) (1.0-1.7) (70.0-80.2)

n is the number of subjects in each group from which themean was obtained. The range of values in each group isshown in parentheses.

dation, plasma acetone oxidation rates of 26 (14-34),23 (10-35), and 16 (10-19) /Imol/m2 per min wereobtained. These values must be regarded as only roughapproximations because steady-state values of plasmaacetone and respiratory CO2 specific activities werenot obtained in these single-injection studies. Never-theless, the results suggest that oxidation of plasmaacetone contributed only a small fraction of the totalCO2 output and that the rate of oxidation remainedfairly constant during the fasting period in spite ofchanging plasma acetone concentrations, pool sizes,and turnover rates.

Incorporation of radioactivity into plasma glucosewas determined in three subjects from each of the threegroups studied. In each subject, the time-coursechanges in plasma glucose specific activity was similarto that shown in Fig. 1, i.e., maximum or near-maximumplasma glucose specific activity was achieved by the6th h after [14C]acetone administration. With the 6-hvalues of glucose and acetone specific activities thepercent of glucose derived from acetone was calculatedfrom the equation: %= ([sp act glucose . 6]/[sp act ace-tone ÷3]) x 100. The average values for the 3-d fastednonobese, obese, and 21-d fasted obese subjects were4.2% (2.6-5.8%), 3.1% (2.8-3.4%), and 11.0% (8.5-13.5%), respectively. These results must also be re-garded as only rough approximations because steady-state plasma acetone and glucose specific activitieswere not achieved in these studies.

In two obese, 3-d fasted subjects, preliminary studieswere done to determine time-course incorporation ofradioactivity from [14C]acetone into plasma lipids andperchloric-acid precipitable protein. After [14C]acetoneadministration, radioactivity in both fractions increasedin a linear manner. After 1 h, plasma lipids contained1-2% of plasma total radioactivity and, by the 6th h,these values had increased to 2-5%. Radioactivity inplasma proteins was 3-4% of plasma total radioactivityafter 1 h and increased to 20-30% after 6 h.

Plasma ketone-body specific activities during [3-14C]-AcAc infusion. To define the relationship among thespecific activities of the three plasma ketone bodiesand to determine the immediate precursor of plasmaacetone, two nonobese subjects were fasted for 3 d andadministered [3-14C]AcAc by primed-continuous infu-sion (2). Fig. 5 shows time-course changes in plasmaacetone, AcAc, and ,-OHB specific activities in one ofthe subjects, the same pattern being also observed inthe other subject. The specific activities of AcAc and,f-OHB were constant but, unlike previous similar stud-ies (2, 16-18), were also equal to each other duringthe entire experimental period. The specific activity ofacetone was at first less than that of the other ketonebodies but, by the 2nd h, the values became equal.This lag period was probably a result of mixing of[14C]acetone derived from the administered ['4C]AcAcwith the existing acetone pool. These results suggestthat plasma AcAc is the immediate precursor of plasmaacetone, and that the lack of isotopic equilibration be-tween AcAc and ,8-OHB previously noted by numerousinvestigators employing [3-'4C]AcAc as the tracer (2,16-18) was probably a result of contamination of AcAcradioactivity by the presence of acetone. However,lack of isotope equilibrium between AcAc and ,-OHB,which has also been reported during infusion of labeled,8-OHB (17), cannot be explained on the basis of theseobservations.

DISCUSSION

Changes in the concentrations and rates of productionand use of plasma AcAc and ,6-OHB during starvation-

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FIGURE 5 Time-course changes in plasma acetone, AcAc,and ,f-OHB specific activities during the primed-continuousinfusion of [3-14C]AcAc.

624 Reichard, Haff, Skutches, Paul, Holroyde, and Owen

induced ketosis in the human have been described (11),but similar data for the remaining ketone-body, ace-tone, have not been reported. The presence of acetonein breath (19-23) and biological fluids (22-24) duringketosis has been documented in the literature. How-ever, lack of satisfactory methods to quantitativelydetermined acetone, and the suspicion that the pres-ence of the compound was solely a result of spontaneousgeneration from AcAc, precluded an active researchinterest in its biological and physiological importance.

Our data show that acetone was present in plasma,urine, and breath of the fasting human in whom ap-preciable concentrations and rates of endogenousproduction and use were observed. The concentrationof plasma acetone in some subjects was equal to orgreater than that of plasma AcAc and also in the rangeof values recently reported in ketotic diabeticpatients (22).

There was a direct relationship between plasma ace-tone concentrations and rates of endogenous produc-tion with highest concentrations and production ratesoccurring in two 3-d fasted nonobese subjects and inobese subjects fasted for 21 d. In the latter subjects,the average rate of plasma acetone production was 137,umol/min. During fasting, maximum AcAc productionrates of about 370 ,umol/min have been reported (11).Thus, conversion of AcAc to acetone could account form(137/370) x 100 = 37% of the AcAc produced inthe fasting human, implying that a significant fractionof AcAc production may not undergo immediateterminal oxidation.

Depending upon the plasma acetone concentration,excretion of the compound in breath and urine couldaccount for =2-30% of the endogenous acetoneproduction rate (Table IV). Conversion of acetone toother biological compounds is, therefore, an importantdisposal mechanism. This is in agreement with previousstudies in rats and humans given acetone orally (14, 15).In our subjects, cumulative 14C02 excretion could ac-count for m5-20% of the administered [I4C]acetone,which suggests that oxidation may play an importantrole in acetone elimination. Similar conclusions fromanimal experiments have been reported by others (25-28). Approximated rates of acetone oxidation repre-sented -50-70% of the production rates in nonobeseand obese 3-d fasted subjects and -25% of the pro-duction rates in 21-d fasted obese subjects. Whetheracetone is oxidized directly or after conversion to othercompounds, such as glucose, cannot be determinedfrom our studies.

The appearance of radioactivity in plasma glucose,lipids, and proteins supports the results of somewhatsimilar studies in animals in which labeled carbon ofacetone was found in cholesterol (25), liver glycogen(26, 28), and various amino acids derived from liverand carcass protein (26,28). Incorporation of radioactiv-

ity from acetone into glucose suggests the possibilityof gluconeogenesis from the compound, or a metabo-lite(s) of the compound, a process for which evidencehas been obtained in rats (28) and ketotic cows (29,30).On the basis of our specific activity data, we have cal-culated that -4- 11% of plasma glucose productioncould theoretically be derived from acetone. The valueof 11% was obtained in 21-d fasted obese subjects inwhomnet splanchnic and renal glucose release rates of=370 ,imol/min might be anticipated (31). Thus, the

rate of glucose production from acetone would be(11/100) x 370 = 40 ,umol/min, requiring 80 ,umol/minglucose equivalents of acetone or 59% of the plasmaacetone production rates measured in these subjects.It is recognized that these calculations are based on anunproven assumption that net glucose synthesis fromacetone can occur in the human. They were done,however, to gain some insight into the possible im-portance of this pathway as a mechanism for acetonedisposal.

With isotope-tracer techniques, production rates ofketone bodies have been measured in animals (16, 17)and in the human (2, 18). In many of these studiesemploying [3-14C]AcAc as the tracer, the calculation ofturnover rates was complicated by the lack of isotopicequilibration between AcAc and f3-OHB. Failure toobtain equilibrium was unexpected and difficult tounderstand. The results of our preliminary studies em-ploying [3-'4C]AcAc infusions suggest that the disequi-librium may have been a result of the presence of un-suspected or incompletely removed [14C]acetone in theAcAc fraction. In these studies, total ketone-body pro-duction rates, calculated with the mean specific ac-tivity of acetone, AcAc, and ,3-OHB, were 310-15%greater than the values obtained with the mean totalketone-body specific activity as previously reported (2).Additional studies are required to confirm theseobservations.

ACKNOWLEDGMENTSThe authors gratefully acknowledge the technical assistanceof Maureen Donohue, Sharon Jarman, Joseph Ottaviano, andthe nursing staff of the Clinical Research Unit.

This work was supported by National Institutes of HealthResearch grants AM-13527 and AM-16102; General ClinicalResearch Centers Branch grant 5 M01RR349; and BiomedicalResearch Support grant 5-S07-RR-05585-11.

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